The present inventors have discovered a substantial body of evidence for the occurrence of overproduction of free radicals, i.e. oxidative injury or oxidative stress, has been implicated is an increasing number and variety of human diseases. These include neurodegenerative diseases, e.g. Alzheimer's and Parkinson's disease, ventricular fibrillation, atherosclerosis, myocardial infarction and arrhythmias, diabetes, etc., by demonstrating overproduction of isoprostanes (IsoPs) and neuroprostanes (NeuroPs). IsoPs and NeuroPs are prostaglandin-like compounds produced by free radical induced peroxidation of arachidonic acid (AA) and docosahexaenoic acid (DHA), respectively. Isoketals (IsoKs) and neuroketals (NeuroKs) are highly reactive γ-ketoaldehydes produced by the IsoP and NeuroP pathways, respectively. IsoKs and NeuroKs rapidly adduct to lysyl residues of proteins and also Exhibit A unique and remarkable proclivity to crosslink proteins.
The present invention encompasses discoveries from studies both in brains from patients with Alzheimer's disease (AD) and an animal model of age-related dementia relevant to AD, ApoE null mice overexpressing human ApoE4.
Thus, an aspect of the present invention is identification of proteins adducted by IsoKs and NeuroKs in brains from patients with AD and the mouse model of AD dementia. Furthermore, the present invention demonstrates the relationship between onset of behavioral abnormalities and the occurrence of oxidative stress and IsoK/NeuroK adduct formation in the mouse model. Thus, the ability of selected antioxidants to suppress oxidative injury and IsoK/NeuroK adduct formation and improve behavioral abnormalities in these mice is determined.
Additionally, another aspect of the present invention is a novel pharmacologic intervention with IsoK scavengers such as pyridoxamine (PM) and salicylamine (SA), for example, that preferentially intercepts and prevents IsoKs/NeuroKs from adducting to proteins in vitro, to prevent IsoK/NeuroK adduct formation and improve behavioral abnormalities in these mice. In this regard, suppression of oxidative stress in general and specifically suppression of IsoK/NeuroK adduct formation is a valuable strategy to mitigate the progression of neurodegeneration and dementia in neurological diseases associated with oxidative stress, such as AD.
Oxidative stress plays a fundamental role in the pathogenesis of such diseases, which leads to the formation of IsoKs and NeuroKs that adduct to and alter the function of critical cellular proteins. This in turn also impairs proteasomal degradation of adducted proteins and inhibits proteasome function, causing neuronal dysfunction and death resulting in dementia.
Summary of several abbreviations used herein: F2-isoprostane (F2-IsoP), F4-neuroprostane (F4-NeuroP), isoketal (IsoK), neuroketal (NeuroK), 4-hydroxynonenal (HNE), Alzheimer's disease (AD), arachidonic acid (AA), docosahexaenoic acid (DHA), amyloid precursor protein (APP), amyloid beta (Aβ), paired helical filament (PHF), neurofibrillary tangles (NFT), pyridoxamine (PM), salicylamine (SA), apolipoprotein E (ApoE), vascular dementia (VaD), dementia with Lewy bodies (DLB), multisystem atrophy (MSA), transgenic (Tg), homocysteine (HCys), liquid chromatography (LC), electrospray ionization (ESI), mass spectrometry (MS), collisional induced dissociation (CID), cerebrospinal fluid (CSF).
Isoketals are the most reactive products of lipid peroxidation heretofore identified. IsoKs adduct almost instantaneously to protein lysine residues and readily induce protein-protein cross-links. In spite of the remarkable reactivity of IsoKs, the present inventors have identified compounds that effectively intercept (scavenge) IsoKs from adducting to proteins.
The IsoK scavengers of the present invention are compounds of the present invention, such as salicylamine (SA), for example, and analogs thereof. The compounds of the present invention prevent cell death in cells exposed to a lethal concentration of a general oxidant—hydrogen peroxide.
As indicated herein, IsoKs are a major mediator of oxidant induced cell injury/death. Additionally, as indicated herein, therapeutic use of the IsoK scavengers of the present invention have beneficial effects in a wide variety of diseases associated with oxidative injury.
Age-related dementias are a major and costly public health problem. The most prominent cause of dementia in the elderly is Alzheimer's disease (AD), but others include vascular dementia (VaD), dementia with Lewy bodies (DLB), and multisystem atrophy (MSA). Remarkable advances have been made towards understanding the genetic basis for early-onset familial AD with the identification of mutations in amyloid precursor protein (APP), and presenilin-1 and presenilin-2 genes. However, these mutations account for only about 0.5% of all AD cases. Furthermore, even these mutations do not result in recognizable disease until well into middle age. The widespread abundance of sporadic late-onset AD, and the relatively late age of “early onset” familial AD suggests that factors other than genetic mutations contribute importantly to the development of AD.
One attractive candidate mechanism contributing to the pathogenesis of AD and other dementias is oxidative stress. A considerable body of evidence has been obtained that supports a potential role for oxidative injury in the pathogenesis of AD. First, levels of biomarkers of oxidant injury are increased in the brain and CSF from patients with AD. Moreover, several known risk factors for AD induce an oxidant stress. In this regard, elevated levels of homocysteine has been identified as an important risk factor for AD and inheritance of the ε4 allele of ApoE has been identified as a risk factor for both sporadic AD and DLB. Both of these risk factors, as well as well as elevated levels of Aβ1-42, have been implicated in promoting oxidative stress.
Lipids are a major target of free radical attack, which leads to lipid peroxidation. A variety of products of lipid peroxidation are formed. The present inventors have focused on the characterization of isoprostanes (IsoPs). IsoPs are prostaglandin-like compounds that are formed non-enzymatically in vivo by free radical-induced peroxidation of arachidonic acid (AA) (C20:4ω6). The first class of IsoPs discovered had a prostaglandin F-type cyclopentane ring structure (F2-IsoPs). Intermediate in the formation of IsoPs are labile bicyclic PGH2-like endoperoxides (H2-IsoPs), which are reduced to form F2-IsoPs. The present inventors have found that thiols catalyze this reduction both in vitro and in vivo. However, this reduction is not entirely efficient, allowing rearrangement of H2-IsoPs in vivo to form E-ring and D-ring IsoPs and also isothromboxanes. The present inventors have also shown that IsoP-like compounds are formed from oxidation of docosahexaenoic acid (DHA) (C22:6ω3). Because DHA is highly enriched in neurons these compounds are termed neuroprostanes (NeuroPs). The impetus for characterizing the formation of NeuroPs derived from the hypothesis that measurement of NeuroPs may provide a uniquely sensitive index of oxidative neuronal injury owing to the fact that DHA is highly enriched in neurons and is much more oxidizable than AA. Analogous to the IsoPs, F4-, E4-, and D4-NeuroPs are formed in brain in vivo.
Evidence for enhanced formation of products of lipid peroxidation in AD is formidable. The present inventors have reported that levels of F2-IsoPs are significantly increased in lumbar cerebrospinal fluid (CSF) from living patients with probable AD and that both F2-IsoPs and F4-NeuroPs are significantly increased in postmortem ventricular CSF from patients with documented AD, compared to levels in aged-matched controls. The finding of increased levels of F2-IsoPs in CSF from living patients with probable AD suggests that oxidant injury is an early event in this disease. The present inventors have also found that levels of F4-NeuroPs are increased approximately two-fold in the hippocampus, superior and medial temporal gyri, and inferior parietal lobe of AD brain compared to aged-matched controls. Importantly, however, no significant differences were found in the cerebellum, an area of the brain that is unaffected by AD pathology.
Lipid peroxidation also generates a number of reactive aldehydes, including 4-hydroxy-2-nonenal (HNE), malondialdehyde (MDA), and acrolein. Interest in these aldehydes derives from the fact that they are reactive molecules that adduct and covalently modify proteins and DNA. Levels of these aldehydes have also been found to be increased in AD. The present inventors discovered a series of γ-ketoaldehydes that are orders of magnitude more reactive than any other known product of lipid peroxidation. Moreover, these compounds exhibit a unique proclivity to crosslink proteins to an extent that is not shared by these other aldehydes. These compounds are formed as rearrangement products of H2-IsoP and H4-NeuroP endoperoxides (FIG. 1). Originally, the present inventors called these products isolevuglandins to emphasize their similarity to the cyclooxygenase-derived γ-ketoaldehydes, levuglandins E2 and D2. However, to these compounds have been subsequently referred to as isomeric ketoaldehydes, shortened to isoketals (IsoKs), to emphasize their chemical structure.
Oxidation of AA in vitro produces both F2-IsoPs and IsoKs in nearly equivalent amounts. However, comparison of the formation of F2-IsoPs and IsoKs in vivo is more complex, because IsoKs rapidly adduct to lysyl residues on proteins. FIG. 2 shows the time course of disappearance of free IsoK and HNE during an incubation with bovine serum albumin, which provides an index of the rate of adduction to the protein. Notably, the IsoK adducted to albumin with extreme rapidity, being essentially complete within a few minutes. In striking contrast, approximately 50% of the HNE still remained in free form after 80 minutes.
The above findings explained why repeated attempts in the past to detect free IsoKs during oxidation of biological systems in vitro, e.g. microsomes, and in animal models of oxidant injury in vivo were unsuccessful because they are rapidly sequestered as protein adducts. Therefore, the present inventors undertook to determine the identity of IsoK lysyl adducts as a basis for development of methodology to detect IsoK and NeuroK adducts in biological tissues in their adducted form. It was found that IsoKs adduct to lysines forming a pyrrole, which readily undergoes autoxidation to form stable lactam and hydroxylactam adducts (FIG. 3). Reversible Schiff base adducts were also identified. Schiff base adducts are formed rapidly and then decline over time, whereas lactam adducts accumulate slowly over time. Lysyl protein adducts of IsoKs and NeuroKs are analyzed by LC/MS/MS following enzymatic digestion of proteins to individual amino acids. Enzymatic digestion of proteins is necessary because the adducts degrade during acid hydrolysis. However, this analysis is further complicated by the proclivity of IsoKs and NKs to induce protein crosslinking, which is resistant to hydrolysis. This is demonstrated following incubation of ovalbumin (OVA) with 10 molar equivalents of IsoK for about 4 hours. This results in the formation of extensive cross-linked high molecular weight oligimers. In striking contrast, no cross-linking was observed following incubation of 10 molar equivalents of HNE with ovalbumin (FIG. 4). This is a dramatic example of the differential effects of IsoKs/NKs and HNE on protein adduction and cross-linking.
Without being bound by theory or mechanism, the above observations show that IsoKs/NeuroKs are the most attractive products of lipid peroxidation heretofore identified as candidates that may be responsible for neuronal injury and protein aggregation in AD and other dementias associated with oxidative stress. The extent to which H2-IsoPs undergo rearrangement depends on cellular efficiency to reduce them. Therefore, the amounts of IsoKs and NeuroKs formed depends not only on the amount of H2-IsoPs formed but also cellular reduction conditions. This is an important consideration when choosing therapeutic interventions to suppress the formation of IsoKs and NeuroKs. In this regard, thiol antioxidants would not only suppress the amount of H2-IsoPs/H2-NeuroPs formed but also effectively reduce them to F2-IsoPs/F4-NeuroPs, thereby preventing their rearrangement to IsoKs/NeuroKs. Therefore, thiol antioxidants would likely be more effective than non-thiol containing antioxidants in reducing the formation of IsoKs/NeuroKs.
Thus, an aspect of the present invention is a method of suppressing the formation of IsoKs/NeuroKs by administering an effective amount of thiol antioxidants.
The present inventors have also demonstrated overproduction of IsoPs in another neurodegenerative disease, including AD and Huntington's disease. Thus, IsoKs and NeuroKs are believed to participate in the oxidative neuronal injury that occurs in these and other neurodegenerative diseases.
Risk factors for AD include increasing age, inheritance of genetic mutations that increase levels of Aβ1-42, inheritance of the ApoE4 allele, vitamin deficiencies that increase levels of homocysteine, and head injury. Without being bound by theory or mechanism, the key commonality of these risk factors is that they cause oxidative stress with attendant IsoK and/or NeuroK overproduction in the hippocampus and surrounding areas of brain, which initiates alterations in protein function leading to cellular dysfunction and death. Using immunohistochemistry with an antibody against IsoK protein adducts, the present inventors have obtained evidence that IsoK adducts in the hippocampus from patients with AD are specifically localized to neurons and neuropil. Importantly, neuronal Isok immunoreactivity is absent in areas of brain from patients with AD that are unaffected by AD pathology (cerebellum) and in the hippocampus from aged-matched controls.
Specific mutations in the amyloid precursor protein (APP) have been characterized in some inherited forms of early-onset AD, particularly in one Swedish family (APPswe). These mutations lead to a substantial increase in the levels of the Aβ1-42 in brain and spinal fluid. Aβ1-42 is toxic to cultured neuronal cells, but the mechanism for this toxicity is still somewhat controversial. One potential mechanism is the production of free radicals, since incubating amyloid peptide with neuronal membranes has been shown to induce lipid peroxidation. There have been conflicting reports regarding the presence of increased amounts of lipid peroxidation products in amyloid deposits. However, soluble Aβ1-42 rather than Aβ1-42 in deposits is probably more capable of inducing lipid oxidation.
Folate, vitamin B6, and vitamin B12 are important cofactors in the homocysteine/methionine conversion cycle. Therefore, deficiencies in these vitamins increase homocysteine levels. Folate, vitamin B6, vitamin B12 deficiencies and hyperhomocysteinemia have been reported to be risk factors for AD. In certain at risk populations, from 10 to 30% of elderly persons may be folate or vitamin B12 deficient. A number of studies link hyperhomocysteinemia to increased lipid peroxidation. The present inventors have recently shown that even small increases in homocysteine levels in normal humans are positively and significantly correlated with plasma concentrations of F2-IsoPs. This finding has since been confirmed independently by Davi et al. One proposed mechanism for the link between homocysteine levels and oxidative stress is the participation of the thiol group in redox cycling of copper, which leads to formation of hydrogen peroxide formation and the formation of highly toxic hydroxyl radicals via Fenton chemistry. This can be markedly accelerated in the presence of cysteine. Homocyteine has also been shown to potentiate Aβ mediated neuronal toxicity.
In addition to the role of vitamin B6 in the homocysteine/methionine cycle, vitamin B6 deficiency itself has been found to be an independent risk factor for AD. This suggests that vitamin B6 may play an additional role in limiting oxidative damage.
The present inventors have discovered that key members of the vitamin B6 family, pyridoxamine (PM), salicylamine (SM), etc., acts to trap reactive aldehydes formed during lipid peroxidation, especially IsoKs/NeuroKs. Effectively sequestering these aldehydes as adducts with PM prevents them from adducting to key cellular proteins. In fact, data demonstrates the efficacy by which pyridoxamine prevents IsoKs from adducting to proteins.
Whereas APPswe mice fed a normal diet do not exhibit neurodegenration, Kruman et. al. recently found that placing APPswe mice on a folate deficient diet enriched with homocysteine induced significant neurodegeneration in the CA3 region of the hippocampus, which did not occur in wild-type mice fed the same diet. The folate deficient/homocysteine enriched diet did not increase amyloid deposition. At present, nothing is known about the combined effects of hyperhomocysteinemia and ApoE4 in vivo on lipid peroxidation, proteasome activity, protein aggregation, or behavioral deficits.
Inheritance of the ε4 allele of ApoE (ApoE4) is associated with poorer cognitive performance with age and is currently the only known genetic risk factor for sporadic AD. Inheritance of ApoE4 may also be a genetic risk factor for (DLB). Homozygosity for ε4 is associated with increased oxidative damage in hippocampal pyramidal cells. In the brain, ApoE protein is produced by astrocytes and microglial cells and then secreted as part of lipoprotein particles. ApoE lipoprotein is recognized and internalized by the LDL receptor-related protein (LRP), which in brain is primarily expressed on neuronal cells, including hippocampal pyramidal neurons. ApoE has been identified immunohistochemically within neurons, where it is proposed to interact with microtubule associated proteins, including tau. ApoE3 lipoproteins induce neurite outgrowth in primary neuronal cultures, whereas ApoE4 lipoproteins do not, a process that requires recognition by LRP. For this reason, ApoE3 appears to enhance the ability of neurons to recover from injury. In keeping with this hypothesis, inheritance of ApoE4 increases risk of neurological deficits following head injury.
While numerous studies have demonstrated that genetic ablation of the mouse ApoE gene increases lipid peroxidation in various tissues, it is important to recognize that the presence of the E4 allele, and not complete deficit of the ApoE gene, is the actual risk factor for AD. Therefore, to study the effect of ApoE4 in vivo requires ablating the endogenous mouse ApoE gene and then inserting human ApoE4 as a transgene. Several variants of ApoE4 mice have been generated by placing the transgene under the control of different promoters, including human glial fibrillary acidic protein (gfap-ApoE4), human apoe (apoe-ApoE4), neuron-specific enolase (nse-ApoE4), mouse Thyl (thy-ApoE4), or the human PDGF-β gene (pdgf-ApoE4). The effect of the transgene is promoter dependent and the appropriate promoter is somewhat controversial. Nonetheless, as ApoE protein is found in both neuron and astroglial cells, the effects of the various transgenes do reveal potential effects in each cell type. Significant dendritic alterations occur in pyramidal neurons of apoe-ApoE4 mice accompanied by poorer performance in Morris water maze tests compared to wild-type or ApoE3 mice. Aged gfap-ApoE4 mice show profound changes in performance on the radial arm test of working memory, but do not have increased levels of senile plaque compared to wild-type mice. Injection of kainic acid at doses that do not induce neurodegeneration in nse-ApoE3 mice results in significant neurodegeneration in nse-ApoE4 mice. Aged thy-ApoE4 and pdgf-ApoE4 mice, but not gfap-ApoE4 mice, show hyperphosphorylation of tau and ubiquitin-positive inclusions. In summary, all ApoE4 mice have some forms of neurological deficit with individual variations in their effects on neurodegeneration and protein aggregation. Because gfap-ApoE4 mice produce ApoE4 in astrocytes, the major source of ApoE production, the present inventors have chosen these mice to study the effects of ApoE4 on lipid peroxidation, protease function, and neurodegeneration.
Currently, it is believed that animal models provide the most effective means to compare temporal alterations in protein function that may result from oxidative neuronal injury with the onset of dementia. However, examination of human post-mortem tissue frames expectations for what alterations in protein function are important in dementia. As discussed below, several cellular functions are altered in AD for which there is in vitro evidence that oxidative injury can induce similar alterations. These include altered protease activity, altered cytoskeletal organization, and altered cholinergic function. Therefore, the present invention allows for the determination whether or not these proteins are adducted by IsoKs and/or NeuroKs in AD and if adduction of these proteins precede the onset of diminished mental function in animal models of dementia.
Aggregated ubiquinated proteins is a prevailing feature of many neurodegenerative diseases and dementias. In AD, these take the form of senile plaques and neurofibrillary tangles. In DLB, these aggregates primarily consist of α-synuclein. The cause of protein aggregation is not well understood, but the presence of ubiquitinated proteins suggests a defect in the ubiquitin/proteasome pathway. Indeed, reduced proteasome activity is a common feature of many neurodegenerative diseases. Reduced chymotrypsin-like and postglutamyl peptidase activity of the proteasome has been found in short post-mortem interval autopsied brains from patients with AD, compared to age and sex-matched controls. The decrease in proteasome activity in AD brain appears to be due to a functional deficiency, rather than decreased proteasomal subunit expression.
Proteins can be degraded by the proteasome via 2 independent pathways, both of which are relevant to oxidized proteins and proteins modified by products of lipid peroxidation. The most common pathway is tagging proteins with ubiquitin by specific E2 and E3 ubiquitinating enzymes. The criteria for recognition and tagging of proteins by E2/E3 is a matter of intense investigation, but remains unknown for many proteins. Only multi-ubiquinated proteins are recognized and degraded by the 26S proteasome. The 26S proteasome consists of two major complexes, the 20S proteasome and either 11S or 19S regulatory subunits. The regulatory subunits recognize and unfold ubiquinated proteins, allowing them to enter into the catalytic core of the 20S proteasome. The 20S proteasome is composed of 7 alpha and 7 beta subunits and has three major protease activities: chymotrypsin-like activity, trypsin-like activity, and post-glutamyl peptidase activity. While the 26S proteasome degrades the majority of proteins, the 20S proteasome can function independently to degrade oxidized and denatured proteins. Proteasome inhibitors such as lactacystin and various peptide-aldehydes act by reacting with the catalytic subunits of the 20S proteasome, and thereby inhibit both 20S and 26S proteasome activity.
The underlying mechanism(s) responsible for impaired proteasome function in AD is unclear, but a unifying link between the occurrence of enhanced oxidant injury and impaired proteasome function is a plausible hypothesis. Exposure of fibroblasts to an oxidative stress in the form of 40% oxygen over a period of 12 wks leads to decreased proteasome activity via the formation and accumulation of aggregated lipofuscin/ceroid. Injection of ferric nitrilotriacetate into mice leads to lipid peroxidation and a transient decrease in proteasome activity. Oxidative stress produces at least three products that have been demonstrated to inhibit proteasome activity: reactive oxygen species, hydroxynonenal (HNE), and IsoKs. Of these, only the IsoKs have effects at submicromolar concentrations.
While reduced proteasome activity might directly account for accumulation of aggregated proteins intracellularly, the converse might also be true, that is that aggregated proteins might inhibit proteasome function. This not only would lead to further accumulation of protein aggregates, but potentially might also induce programmed cell death. The latter notion is supported by the findings that synthetic proteasome inhibitors induce apoptosis in neurons, probably through the accumulation of proapoptotic signals such as p53. In summary, lipid peroxidation, protein aggregation, and inhibition of proteasome function may be casually linked and intertwined, which in concert could lead to neurodegeneration.
In addition to the proteasome, another protease whose activity has been shown to be reduced by approximately 50% in AD is insulin degrading enzyme (IDE). IDE degrades not only insulin but also Aβ1-42 peptide. Very recent studies have demonstrated that genetic ablation of IDE increases brain Aβ1-42 levels. Addition of insulin to IDE inhibits amyloid degradation by IDE, and increases secreted levels of Aβ1-42 in neuro2A cells. IDE strongly associates with the proteasome and addition of insulin to IDE also inhibits proteasomal chymotrypsin-like activity through an unknown mechanism. Because of this relationship, alterations in IDE activity in vivo might also effect proteasomal activity or vice versa. If IsoK or NeuroK adduction of IDE inhibits its activity, it is conceivable that Aβ1-42 levels would increase and proteasome activity would decrease, accounting for two major findings in AD. For this reason, determining whether IDE activity decreases in animal models of AD and whether changes in activity correlate with IDE adduction by IsoKs/NeuroKs is an aspect of the present invention.
Another major protease activity in AD in calpain, which is increased in the disease. Calpain cleavage of the precursor of cdk5 activates cdk5, which phosphorylates tau. Calpain also cleaves tau, but not phosphorylated-tau or paired helical filament (PHF)-tau. Therefore, increased calpain activity could lead to increased phosphorylation of tau, a prerequisite for PHF-tau formation. Interestingly, oxidative injury and amyloid β both have been shown to increase calpain activity, which potentially may link levels of amyoid β and oxidative stress to PHF-tau formation.
Neurofibrillary tangles (NFT), which are comprised primarily of ubiquinated aggregates of PHF-tau, are a defining feature of AD. PHF-tau is phosphorylated at several well-characterized sites. In vitro, tau promotes microtubule assembly, stabilizes cellular microtubules, affects their dynamic behavior, and may play an important role in regulating microtubule interactions with membranes. Formation of the abnormal tau aggregates, PHF-tau and NFT, therefore may lead to the loss of normal neuronal structure and function. The underlying process that leads to the formation of PHF-tau and NFT formation in AD is currently unknown. One plausible hypothesis is that products of lipid peroxidation modify phosphorylated tau, preventing its degradation and thereby facilitating its aggregation. In support of this hypothesis, incubation of phosphorylated tau with products of lipid peroxidation, e.g. HNE, can induce the epitope for Alz50, an antibody that recognizes an AD-specific conformational epitope on PHF-tau. Furthermore, HNE inhibits dephosphorylation of tau in primary rat hippocampal cells. Immunohistochemical studies have also localized both immunoreactive HNE and acrolein to neurofibrillary tangles. As mM concentrations of HNE are required to induce the Alz50 epitope on tau, more reactive products of lipid peroxidation, such as IsoKs and NeuroKs, may be more attractive candidates involved in initial modification of tau in AD. Important in that regard is that we have found that adduction of IsoKs to tau does induce the Alz50 epitope.
The cellular function of microtubules include axonal and dendritic growth and support of sympatic activity. Ultrastructural analysis of cortical neurons suggest abnormalities of the cytoskeleton in AD. The underlying cause of this disruption is unknown, but products of lipid peroxidation, e.g. HNE, have been hypothesized to play a role. Incubation of mouse neuroblastoma Neuro2A cells with HNE results in disruption of microtubule organization and inhibition of neurite outgrowth. When cytoplasmic proteins from these cells were examined, HNE adducts were found on both tubulin and tau protein. Since one of the major functions of tau protein is to organize tubulin filaments, these experiments suggest that adduction of these proteins is responsible for microtubule disruption. Presumably, adduction of tubulin by IsoKs or NeuroKs would similarly disrupt microtubule organization.
Impairments in cholinergic neurotransmitter systems of the basal forebrain are a hallmark of Alzheimer's disease pathophysiology. The deficit in acetylcholine synthesis results from reduction in choline acetyltransferase activity. Reduction of acetyltranferase activity is greater than the loss of cholinergic neurons. Therefore, neuronal death may be a result of, not the cause of, the reduction in acetytransferase activity. Interestingly, exposure of neuronal cells in culture to HNE or subjecting them to an oxidative stress has been shown to decrease choline acetyltransferase activity. However, in the case of the former, it is not known whether this effect is a result of adduction of the enzyme by HNE or impairment of transcription of the choline acetyltransferase gene.
There are two Nerve Growth Factor (NGF) receptors, trkA and P75NGFR. Both NGF receptors localize to cholinergic neurons in the hippocampus and NGF receptors are depleted in AD. Although trkA mRNAs are decreased in AD, loss of NGF binding appears to precede the loss of TrkA immunoreactivity and neuronal loss, suggesting that at least some of the loss of receptor function is due to receptor modification. Exposure of neuronal cultures to hydrogen peroxide or Aβ1-42 reduces the level of trkA protein, suggesting that oxidative injury may play a role in receptor loss.
In summary, there are a number of known proteins that are thought to play important functions in either the progression of AD or protection against the disease and whose function is altered in AD. Therefore, aspects of the present invention include the determination that these proteins in AD brain and brain from an animal model of a risk factor for AD are adducted by IsoKs and/or NeuroKs.
Purely for exemplary purposes, the following non-inclusive aspects of the present invention are discussed. These embodiments should not be considered as limiting, and other embodiments as would be obvious to one of ordinary skill in the are to be considered as part of this invention and not departures therefrom.
A first aspect of the present invention is to provide a method for assessing the formation of IsoK and NeuroK adducts quantitatively and qualitatively in AD brain.
A second aspect of the present invention is a method to assess potential causative factors involved in age-related dementia. This may include the use of ApoE null mice transgenically expressing human ApoE4 by evaluating the brain levels of F2-IsoPs, F4-NeuroPs, and levels and distribution of IsoK and NeuroK adducts.
A third embodiment of the present invention is a method to determine the role of oxidative stress/injury in general and specifically the role of IsoK/NeuroK adduct formation by using pharmacologic interventions that include effective amounts of at least one of antioxidants, Tempol, and lipoic acid.
A fourth aspect of the present invention is a method of identifying the proteins adducted by IsoKs and NeuroKs in the hippocampus of brains from patients with AD and in ApoE4 Tg mice fed a normal or folate deficient/homocysteine enriched diet. This may include determining whether there is enhanced adduction of the following proteins by IsoKs and NeuroKs: tau, tubulin, proteasome subunits, insulin degrading enzyme, acetylcholine acyltransferase, ApoE, and neuronal growth factor receptors.
One embodiment of the present invention is a method of treating and/or preventing oxidative damage that comprises administering an effective IsoK/NeuroK adduct formation suppressing amount of a phenolic amine compound and/or a pyridoxamine compound.
Another embodiment of the present invention is a method of preventing myocardial damage that comprises administering a damage preventing effective amount of phenolic amine compound or a pyridoxamine analog or salicylamine analog.
Another embodiment of the present is a method of preventing cardiac sodium channel dysfunction, inactivation, and/or blocking that comprises administering an effective amount of a phenolic amine compound and/or a pyridoxamine analog or salicylamine analog.
Another embodiment of the present invention is a method of preventing or treating ventricular fibrillation and/or arrhythmias that comprises administering an effective amount of a phenolic amine compound and/or a pyridoxamine analog or salicylamine analog.
Yet another embodiment of the present invention is a method of preventing or retarding the progression of neurodegenerative disease that comprises administering an effective oxidative stress preventing or decreasing amount of a phenolic amine compound and/or a pyridoxamine or salicylamine compound. The neurodegenerative disease of this or other embodiments may include, but is not limited to, Parkinson's disease, Alzheimer's disease, Huntington's disease and/or dementia.
Another embodiment of the present invention is a method of preventing or retarding the progression of oxidative stress associated with vascular dementia or stroke, comprising administering an effective oxidative stress preventing or decreasing amount of a phenolic amine compound and/or a pyridoxamine or salicylamine compound.
Any of these embodiments may include the use of a pyridoxamine, salicylamine, tyrosine compound or an analog thereof. Examples of these compounds or analogs include, but are not limited to, compounds selected from the formula:
wherein:    R is N or C;    R2 is independently H, substituted or unsubstituted alkyl;    R3 is H, halogen, alkoxy, hydroxyl, nitro;    R4 is H, substituted or unsubstituted alkyl, carboxyl; or analogs thereof.